The neurodegenerative disease amyotrophic lateral sclerosis (ALS) and acquired immunodeficiency syndrome (AIDS) are diseases with quite different etiologies and symptoms yet they share an important connection to the human RNA debranching enzyme Dbr1. The main cellular role of Dbr1 is to help recycle intron RNAs following pre-mRNA splicing by cleaving the 2'-5' bond at intron lariat RNA branch points. Interestingly, loss of Dbr1 activity inhibits replication of Human Immunodeficiency Virus (HIV), the retroviral agent of AIDS. Surprisingly, it was recently discovered that accumulation of intron RNA lariats resulting from loss of Dbr1 activity also alleviates toxicity caused by cytoplasmic aggregation of the ALS associated RNA binding protein TDP-43. A consequence of these findings is that Dbr1 inhibitors have potential as anti-ALS and anti-HIV/AIDS drugs. The long-term goal of our research is to innovate novel Dbr1-based therapeutic strategies for treating ALS and HIV infection. The objective of this application is to develop a high-throughput phenotypic screen for Dbr1 inhibitors. Our central hypothesis is that a phenotypic screen using the budding yeast Saccharomyces cerevisiae can identify human Dbr1 inhibitors. Taking advantage of a novel yeast lariat intron reporter strain, research will focus on two specific aims. Aim 1 is to identify yeast Dbr1 inhibitors. For this aim a pilot high-throughput phenotypic screen of small molecule libraries will be performed, assaying for the intron RNA lariat signal in our yeast reporter strain as a proxy for Dbr1 activity. Hit compounds will be validated with independent assays for intron RNA lariat accumulation and Dbr1 enzyme activity. Select hit compounds will be further assessed for their effects on yeast models for ALS and HIV. Aim2 will be to identify human Dbr1 inhibitors by testing yeast Dbr1 inhibitors on human cells and the human Dbr1 enzyme. Follow up studies will examine the effects of human Dbr1 inhibitors on TDP-43 cytotoxicity and HIV-1 replication in human cells. The significance of the work is it will create the means to identify lead compounds that have potential as completely novel treatments for ALS and HIV/AIDS.